Abstract

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and ∼60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation (V600EB-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, ∼70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting V600EB-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound–mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting V600EB-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting V600EB-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of V600EB-Raf and Akt3 led to a cooperatively acting ∼65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting V600EB-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas. [Cancer Res 2008;68(18):7638–49]

melanoma

ultrasound

B-Raf

nanoliposomes

siRNA

Introduction

Melanoma is a cancer of pigmented skin cells called melanocytes residing at the epidermal-dermal junction (
1). Most genetic alterations promoting development of early melanomas are yet to be identified. One frequent change occurring in ∼90% of normal moles is a T1799A mutation of B-Raf in the mitogen-activated protein (MAP) kinase pathway (
2–
5). Approximately 60% of advanced-stage metastatic melanomas also contain the activating V600E mutation leading to 10.7 times higher activity than occurs in normal cells, thereby promoting cellular proliferation (
2–
5).

A second frequent alteration promoting melanoma development is increased Akt3 activity in ∼70% of melanomas (
6). Akt3 is activated through loss of the PTEN phosphatase and increased gene copy number, leading to deregulation of apoptosis, thereby promoting chemoresistance (
6,
7). Targeting V600EB-Raf or Akt3 signaling decreases melanoma tumor development, and simultaneous inhibition can lead to cooperatively acting tumor inhibition (
6,
8,
9). Thus, agents specifically targeting V600EB-Raf and/or Akt3 could have significant potential to treat moles, early melanocytic lesions, or skin metastases in which these proteins are deregulated.

Inhibition of B-Raf or Akt3 using small interfering RNA (siRNA) specifically designed to inhibit V600EB-Raf but not wild-type B-Raf or Akt3 but not Akt1 or Akt2 protein expression can effectively decrease melanoma tumor development in mice (
6,
8,
10). Because these agents target specific genes, there would be fewer potential side effects than with drugs inhibiting both mutant and wild-type B-Raf or all three Akt isoforms (
6,
8,
10). Introduction of siRNA in cultured cells is easily accomplished using standard transfection methods (
11–
13). However, its use as a targeted chemopreventive or therapeutic agent is limited due to degradation in animal systems (
14,
15).

Encapsulating siRNA into nanoliposomes can be used for protection and systemic delivery, leading to tumor inhibition (
16–
18). In contrast, topical administration on skin leads to uptake confined to the upper epidermal layers without complete skin penetration (
19). This is due, in part, to skin architecture, with epidermis and dermis protected by a surface keratinized layer (
19,
20), which prevents most topically applied agents from reaching early melanocytic lesions developing at the epidermal-dermal junction or from reaching locally invasive cutaneous melanoma metastases in the skin dermis (
21). One promising strategy for delivery through the keratinized layer is the use of low-frequency ultrasound to permeabilize the skin, enabling passage of macromolecules, including antisense oligonucleotides without significant skin damage (
21–
26). Combining the protective qualities of nanoliposomes with the permeabilizing activities of low-frequency ultrasound is a novel unexplored strategy for delivering siRNA into melanocytic lesions present in skin to prevent or treat cutaneous melanoma.

This study details the development of a unique nanoliposomal-ultrasound–mediated approach for delivering siRNA targeting V600EB-Raf and Akt3 protein expression into melanocytic lesions present in skin to inhibit early lesion development or prevent cutaneous melanoma metastasis.

Liposome composition, characterization, and siRNA loading. Nanoliposomes were created by combining 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] [DSPE-PEG(2000); Avanti Polar Lipids] in chloroform at a 4.75:4.75:0.5 molar ratio (
28,
29). siRNA and nanoliposomes complexing occurred at specific weight ratios of 1:5, 1:10, and 1:15 for 0.5 to 24 h. A quasielastic light scattering system (Malvern Nanosizer, Malvern Instruments) was used to measure the particle diameter of nanoliposomes ± siRNA 1 d after preparation. Nanoliposomal loading of siRNA was measured by mixing fluorescent, Alexa Fluor 546–tagged siRNA (Qiagen) with nanoliposomes at specific weight ratios (1:5, 1:10, 1:15) and complexing at room temperature for 0.5, 3, or 6 h. Loading was considered complete when no free siRNA was evident with complex remaining in well of a 2% agarose gel. To measure liposome-mediated protection of siRNA, nanoliposomes complexed with siRNA overnight were treated with 50% FBS for 10, 30, 60, 180, or 360 min. Following FBS treatment, half of the complex at each time point was treated with 0.5% SDS for 10 min at 37°C to disrupt the complexes and release free siRNA. siRNA alone was also digested with 50% FBS at each time point to serve as a control. All samples were run on a 2% agarose gel at 100 V for 20 min and visualized under UV.

Generation of skin containing melanocytic lesions. To create skin in a culture dish, human fibroblasts were trypsinized and resuspended in 10% reconstitution buffer, 10% 10× DMEM (Mediatech), 2.4 μL/mL of 10 mol/L NaOH, and 80% collagen I (Becton Dickinson) at a concentration of 2.5 × 105/mL on ice (
32). Mixture was then aliquoted into 6- or 12-well plates and incubated at 37°C for 3 h. E-medium was added to each well to equilibrate the dermal matrix (
32). After 2 d of growth, keratinocytes and melanoma cells (WM35-GFP or UACC 903-GFP) were trypsinized and resuspended at a 1:10 ratio of melanoma cells (nucleofected or untreated) to keratinocytes in E-medium. One milliliter of cell suspension was added to each well on top of the dermal layer. Following 2 d of growth, reconstructed skin was transferred onto wire grids and fed via diffusion from E-medium below the platforms.

Ultrasound transducer design and calibration. The cymbal transducer is a novel thin flextensional transducer capable of producing very low frequencies (
33,
34). It has a compact, lightweight structure with an adjustable resonance frequency. Caps on the lead zirconate-titanate ceramic contain a shallow cavity beneath the inner surface. The fundamental mode of vibration is flexing of the end caps caused by radial motion of the ceramic. Therefore, the overall displacement of the device is a combination of the axial motion of disk plus radial motion amplified by the end caps (
23,
24). A radiofrequency signal was generated by a frequency pulse/function generator (Agilent 32250A) and amplified using an amplifier (model 40A12, Amplifier Research). Electrical impedance of the array was tuned to the output impedance of the amplifier by an external inductor-capacitor tuning network. Pulse period, duty cycle, and exposure time of radiofrequency signal from the frequency generator were monitored using an oscilloscope (Tektronix 2213A). For experiments, signal generator operated at 20 kHz with pulse duration of 200 ms and pulse repetition period of 1 s (i.e., 20% duty cycle); the amplifier gain was set to 50 dB. Pulsed ultrasound was used to avoid heat generation, which could damage array or skin.

Ultrasound treatment and analysis of damage, nanoliposomal-siRNA uptake, and tumor inhibition in reconstructed skin. Skin reconstructs were treated with ultrasound at a frequency of 20 kHz and duty cycle of 20% for 20 min by removing media and submersion in PBS (
26). The nanoliposomal-siRNA complex (1 μmol/L) was then applied topically and allowed to soak into the skin. Skin damage was assessed following ultrasound treatment, addition of ghost liposome, and fixation in 4% paraformaldehyde at 4°C overnight. H&E-stained paraffin-embedded cross sections were examined for skin damage by microscopy. Skin reconstruct uptake studies involved treating with nanoliposomes containing 1 μmol/L Alexa Fluor 546–tagged siRNA or ghost nanoliposomes and uptake was analyzed using stereo fluorescence microscopy 1 h later. Skin reconstructs were treated with ultrasound followed by treatment with nanoliposomes containing B-Raf siRNA or ghost nanoliposomes on alternate days from days 10 to 21. Skin reconstructs from all experiments were fixed in 4% paraformaldehyde at 4°C overnight. After fixation, skin was stored in 0.5 mol/L EDTA (pH 8.0; Fisher Scientific). The total average area occupied by green fluorescent protein (GFP)–tagged tumor nodules present in four to six skin images per skin was used to quantify differences between treatment groups using the IP Lab software.

Ultrasound and siRNA-nanoliposomal complex treatment of tumors. UACC 903-GFP cells (1 × 106) were injected s.c. into the right flank of nude mice. After 24 h, mice were ultrasound treated for 15 min followed by topical application of 25-μg nanoliposomal-siRNA (siMutB-Raf, siAKt3, or siScrambled) complex on alternate days. For combination nanoliposomal V600EB-Raf and Akt3 siRNA studies, mice were treated with the same regimen as the single agents. Single agents were compensated with extra liposomes so that equivalent amounts were used. Before ultrasound treatment, mice were anesthetized using 2:1 ketamine/xylazine, and area was sterilized with alcohol pads. Aquaflex Gel Pads (Parker Laboratories, Inc.) served as an ultrasound standoff. Ultrasound was administered for 15 min at a frequency of 20 kHz and duty cycle of 20% (
26). Nanoliposomal-siRNA was applied to ultrasound-treated area overlying the tumor for penetration into skin.

Toxicity analysis in mice. To establish toxicity associated with ultrasound plus siRNA-nanoliposomal complex, nude mice were ultrasound treated for 15 min followed by topical addition of nanoliposomal-siRNA (siMutB-Raf, siAkt3, siScrambled, or siMutB-Raf + siAkt3) complex on alternate days. Mice were weighed on alternate days to ascertain toxicity. Following euthanization on day 23, blood was collected from mice and serum separated to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, and alkaline phosphatase. Livers were fixed in formalin, sectioned, and stained with H&E to assess liver morphology changes.

Statistical analysis. One-way or two-way ANOVA followed by appropriate post hoc test (Tukey's or Bonferroni) or t test was used to ascertain whether significant differences occurred between groups. Differences were considered significant at P < 0.05. Combination index was calculated using the Chou-Talalay method with CalcuSyn software to establish mechanistic basis for cooperative inhibition mediated by siRNA targeting V600EB-Raf and Akt3 (
35).

Results

Development of stable cationic nanoliposomes that load siRNA. Because nanoliposomes have been reported as potential siRNA delivery vehicles, a number of possible formulations were evaluated and a novel DOTAP/DOPE/DSPE-PEG(2000) ratio of 4.75:4.75:0.5 was found to efficiently load siRNA. Loading was measured by adding Alexa Fluor 546–tagged siRNA to nanoliposomes at ratios of 1:5, 1:10, or 1:15 (siRNA/liposome, by weight) and complexed for 0.5, 3, or 6 hours. Measuring free fluorescently tagged siRNA present in the gel or as a siRNA-liposome complex retained in the gel well was used to directly assess siRNA loading. A 1:5 ratio following a 0.5-hour incubation showed the presence of both loaded and unloaded siRNA, which was in contrast to the 1:10 and 1:15 ratios where siRNA remained in gel wells, indicating complete complexing with nanoliposomes (
Fig. 1A, top left
). Because maximal loading occurred following 30-min incubation at a 1:10 ratio, this siRNA-nanoliposomal complex formulation was used for subsequent experiments. The diameter of siRNA-liposome complexes 1 day after preparation was measured by dynamic light scattering, showing an average diameter of ∼50 nm with a range of 34 to 67 nm (
Fig. 1A, top right). Ghost nanoliposomes had a similar diameter averaging ∼48 nm with a range of 32 to 64 nm. Thus, stable nanoliposomes were generated that loaded siRNA.

Characterization of cationic siRNA-liposome complexes. A, loading of fluorescently tagged siRNA into nanoliposomes. Fluorescently tagged siRNA was complexed with cationic nanoliposomes at ratios of 1:5, 1:10, or 1:15 for 0.5, 3, or 6 h and run on a 2% agarose gel to determine loading efficiency. Maximal loading was reached at a 1:10 ratio following a 0.5-h incubation (top left). siRNAs at a 1:10 ratio with nanoliposomes were sized using dynamic light scattering and similar size ranges were observed for ghost or nanoliposomes loaded with siRNA (top right). siRNA protection by cationic nanoliposomes was measured by complexing fluorescent siRNA with nanoliposomes overnight followed by exposure to serum for 10, 30, 60, 180, or 360 min. Free fluorescent siRNA alone was used as a control (bottom left and middle). Release of siRNA in nanoliposomes was accomplished by collapsing serum-treated siRNA-nanoliposomal complexes with SDS to release free siRNA, which was then run on an agarose gel (bottom right). B, siRNA-nanoliposomal complex is taken up into normal cells. Uptake of siRNA-nanoliposomal complex into normal cells was measured by adding fluorescently tagged siRNA-nanoliposomal complex (200 nmol/L) to fibroblasts, keratinocytes, and melanocytes for 3 h followed by fixation and imaging by fluorescence microscopy (magnification, ×400). Ghost nanoliposomes lacking fluorescent siRNA were used as a control. C, siRNA-nanoliposomal complex is taken up into the cytoplasm of cells and is not merely surface bound. siRNA-nanoliposomal complex localization in cells following treatment with siRNA-nanoliposomal complexes (100 nmol/L) was ascertained by exposing 1205 Lu melanoma cells for 3 h, after which cells were trypsinized to remove surface-bound complex and replated overnight onto coverslips. Cells were fixed and imaged by fluorescence microscopy (magnification, ×400). D, ghost liposome or siRNA-nanoliposomal complex exerted negligible toxicity on normal or cancer cells. Cellular toxicity of ghost liposome and siRNA-nanoliposomal complex was measured by adding nanoliposomes (12.5, 25, and 50 μmol/L) to fibroblasts, keratinocytes, melanocytes, or melanoma cells for 24 h followed by MTS assay analysis. Untreated cells (−) served as controls for comparison. Columns, mean; bars, SE.

siRNA in nanoliposomes is protected from serum degradation. To evaluate the protective effects conferred by nanoliposomes on siRNA, fluorescently tagged free siRNA or siRNA-liposome complexes were exposed to 50% FBS, which is rich in RNA degrading factors, for 10, 30, 60, 180, or 360 minutes. Partial degradation of free siRNA was observed after 10 minutes and complete degradation was observed at 60 minutes (
Fig. 1A, bottom left). In contrast, siRNA-nanoliposomal complexes remained in gel wells at all time points, indicating protection from degradation (
Fig. 1A, bottom middle). Half of the original sample was treated with 0.5% SDS for 10 minutes, disrupting the complex and releasing siRNA, which was then run on an agarose gel. Similar levels of free siRNA were observed at all time points, indicating protection by the complex (
Fig. 1A, bottom right). Thus, siRNA in nanoliposomes was protected from degradation by external factors.

Nanoliposomal-siRNA complex is nontoxic and taken up into the cytoplasm of normal as well as cancer cells. To determine nanoliposomes mediated cellular uptake of siRNA, fibroblasts, keratinocytes, and melanocytes were treated with ghost nanoliposomes or nanoliposomes containing Alexa Flour 546–tagged siRNA (50, 100, or 200 nmol/L). Fluorescence microscopy showed red Alexa fluorescence in ∼100% of all cells, indicating uptake of nanoliposomal-siRNA (
Fig. 1B). To verify that siRNA was taken into the cytoplasm of cells, 1205 Lu cells were treated with 100 nmol/L fluorescein-tagged siRNA-nanoliposomal complex or ghost nanoliposomes. Cells were then trypsinized to remove cell surface–bound nanoliposomes and replated overnight followed by fixation (
36). Fluorescence microscopy showed the presence of green fluorescein–tagged siRNA in cytoplasm of cells surrounding a nuclear shadow, indicating that nanoliposomal-siRNA was internalized (
Fig. 1C). In contrast, no green fluorescence was observed in cells treated with ghost nanoliposomes. Next, toxicity mediated by ghost nanoliposomes was examined in fibroblast, keratinocyte, melanocyte, or 1205 Lu melanoma cells. Cells were treated with increasing concentrations of ghost nanoliposomes (12.5, 25, and 50 μmol/L) for 24 hours and analyzed by MTS assay (
37). Compared with untreated cells, no significant decrease in cellular viability was observed, indicating that ghost nanoliposomes were not toxic at the concentrations examined (
Fig. 1D). The toxicity of siRNA-nanoliposomal complex was next examined in 1205 Lu cells, comparing untreated cells to those treated with increasing concentrations of complex loaded with scrambled siRNA (
Fig. 1D). No change in cell viability was observed, indicating negligible toxicity. Thus, the siRNA-liposome complex was nontoxic and taken up into the cytoplasm of normal and cancer cells.

siRNA targeting V600EB-Raf specifically decreases mutant, but not wild-type, protein expression in cells. siRNA can be designed overlapping the T1799A mutation site of V600EB-Raf, which has potential to selectively decrease the expression of mutant, but not wild-type, protein (
8,
10,
30). To verify the specificity of siRNA targeting V600EB-Raf, 100 pmol (1 μmol/L) was nucleofected into melanoma cells containing mutant (UACC 903) or wild-type protein (C8161.Cl9). Western blot and densitometric analysis of cellular protein lysates showed that siRNA designed specifically against V600EB-Raf reduced protein levels by ∼50% in mutant UACC 903 (
Fig. 2A
) but not in wild-type B-Raf–expressing C8161.Cl9 cells (
Fig. 2B). In contrast, siRNA designed to sequences present in both mutant and wild-type B-Raf decreased protein expression by ∼75% in both cell lines (
Fig. 2A and B). Similarly, siRNA that specifically targets Akt3 but not Akt1 or Akt2 has been reported, which can effectively decrease melanoma tumor development (
6). Thus, siRNA designed to specifically inhibit mutant V600EB-Raf or Akt3 reduces the expression of mutant but not wild-type B-Raf protein and Akt3 but not Akt1 or Akt2, respectively (
6).

siMutB-Raf decreases protein expression of V600EB-Raf. A and B, siRNA can be designed to decrease V600EB-Raf expression but not normal protein expression. To verify specificity of siMutB-Raf for decreasing expression of mutant but not wild-type protein, melanoma cells containing mutant (UACC 903; A) or normal (C8161.Cl9; B) B-Raf protein were nucleofected with buffer, siScrambled, siC-Raf, siWTB-Raf, or siMutB-Raf siRNA; protein lysates were harvested 48 h later and analyzed by Western blot analysis for B-Raf and C-Raf knockdown. Erk2 served as a loading control. C, siMutB-Raf-nanoliposomal complex decreased the expression of V600EB-Raf protein in cells. 1205 Lu cells were exposed to siMutB-Raf or siScrambled-nanoliposomal complex; protein lysates were harvested at 18 and 32 h and analyzed by Western blot analysis. Erk2 served as a control for protein loading. Columns, mean; bars, SE. D, duration of V600EB-Raf protein knockdown following exposure to siRNA targeting mutant protein is beyond 8 d. Cells were nucleofected with C-Raf or V600EB-Raf siRNA and replated in culture dishes; protein was harvested 2, 4, 6, and 8 d later to measure duration of protein knockdown. Untreated cells or cells nucleofected with siRNA targeting C-Raf served as controls.

siRNA targeting V600EB-Raf can be loaded into nanoliposomes, delivered into cells, and decrease expression of mutant protein. Having validated the specificity of siRNA targeting V600EB-Raf and Akt3, 1 μmol/L siRNA was loaded into nanoliposomes, and protein knockdown measured in 1205 Lu cells containing V600EB-Raf in three independent experiments by densitometric analysis and Western blotting (
Fig. 2C). Examples of protein knockdown at 18 and 32 hours following a single treatment showed a 50% to 60% decrease of V600EB-Raf protein compared with cells treated with scrambled siRNA-nanoliposomal complex (
Fig. 2C). Typically, protein knockdown ranged from 25% to 60%, indicating some variability, but knockdown was reproducibly observed. Duration of knockdown following a single siRNA treatment was measured by introducing siRNA into cells and using Western blotting to measure protein knockdown 2, 4, 6, and 8 days later. Western blots were densitometrically scanned and knockdown of B-Raf protein was compared with control cells nucleofected with siRNA targeting C-Raf. A consistent ∼60% decrease in protein expression was observed through day 8 (
Fig. 2D). Thus, siRNA specific to V600EB-Raf can be loaded into cationic nanoliposomes, delivered into melanoma cells, and decrease protein expression by 25% to 60% following a single treatment.

Ultrasound treatment of laboratory-generated skin containing melanocytic lesions enables delivery of siRNA-nanoliposomal complex into tumors. Skin forms a natural barrier that prevents uptake of agents including lipids and siRNA (
19,
21). Therefore, skin was permeabilized before addition of siRNA-liposomal complex using a lightweight four-cymbal array ultrasound developed originally to deliver insulin through skin and into the blood stream (
Fig. 3A, left
; refs.
23–
26). A representative exposimetry analysis showed a Gaussian-shaped peak field intensity with an overall spatial peak-temporal intensity of 50.6 ± 1.5 mW/cm2 (
Fig. 3A, right). Possible skin damage by ultrasound was evaluated on reconstructed skin treated with ultrasound for 20 minutes followed by addition of topically added ghost liposome for 1 hour followed by fixation. H&E-stained skin sections comparing untreated control to ultrasound- and ghost liposome–treated skin showed no detectable damage (
Fig. 3B). Next, skin penetration by siRNA-nanoliposomal complex following ultrasound treatment was measured. Skin was ultrasound treated for 20 minutes followed by topical addition of liposome loaded with 1 μmol/L Alexa Fluor 546–tagged siRNA and, 1 hour later, fixed in 4% paraformaldehyde followed by analysis using fluorescence stereomicroscopy. Top-down views of the skin showed uptake of red Alexa Fluor 546–tagged siRNA in both keratinocytes and UACC 903-GFP melanoma cells (
Fig. 3C). Cross sections of skin showed the presence of red Alexa Fluor 546–tagged siRNA in melanoma cells located in both epidermis and epidermal-dermal junction (
Fig. 3D). Similar results were seen in skin reconstructs containing WM35-GFP (data not shown). Thus, permeabilizing skin using ultrasound before topical application of siRNA-nanoliposomal complex enabled delivery of siRNA to melanocytic lesions located within skin.

Ultrasound treatment permeabilizes skin, enabling melanocytic lesions to take up siRNA-nanoliposomal complexes. A, ultrasound assembly. A lightweight, low-profile ultrasound array was constructed using four-cymbal transducers, which were connected in parallel and encased in polymer. The temporal peak intensity was determined in a spatial plane 1 mm from the face of the transducer for exposure conditions. B, ultrasound treatment does not damage skin. Laboratory-generated skin was ultrasound treated followed by addition of ghost liposome, skin sectioned, and H&E stained. No changes in cellular structure or skin morphology were observed compared with untreated control skin (magnification, ×200). C and D, following ultrasound treatment of skin, siRNA-nanoliposomal complex is taken up by melanocytic lesions in the epidermis and at the epidermal-dermal junction. Following 20-min ultrasound treatment, fluorescent siRNA-nanoliposomal complex or ghost nanoliposomes were applied topically onto reconstructed skin. One hour later, skin was fixed and analyzed by fluorescence microscopy looking down at the skin (magnification, ×4; C) or by cross sections (magnification, ×400; D). Fluorescence (red) indicating the presence of siRNA-nanoliposomal complex was evident in melanocytic lesions in both epidermis and at epidermal-dermal junction (arrows).

Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complex inhibits melanocytic lesion development in reconstructed skin. A, schematic showing treatment regimen. Beginning on day 10 and on alternate days thereafter up to day 20, reconstructed skin was treated with ultrasound for 20 min followed by topical administration of siMutB-Raf-nanoliposomal complex (100 pmol) or ghost nanoliposomes. B and C, ultrasound followed by addition of siMutB-Raf-nanoliposomal complex decreases melanocytic lesion development in skin. Reconstructed skin containing UACC 903-GFP or WM35-GFP cells was treated with ultrasound for 20 min followed by topical administration of siMutB-Raf-nanoliposomal complex on alternate days from days 10 to 20. Skin was harvested on day 21, and the average area occupied by GFP-tagged tumors was calculated for each group. Ultrasound treatments followed by exposure to ghost nanoliposomes served as a control. Columns, mean; bars, SE.

Mechanistically, siRNA targeting V600EB-Raf decreases the proliferative potential of melanoma cells by inhibiting MAP kinase signaling in laboratory-generated skin. To establish the mechanistic basis by which siRNA targeting V600EB-Raf inhibited melanoma development, siRNA was introduced into GFP-tagged WM35 or UACC 903 cells, which were seeded into the epidermis of reconstructed skin. Ten days later, skin was fixed and fluorescence microscopy was used to quantify the average area occupied by GFP-expressing tumor nodules. Compared with cells nucleofected with buffer, scrambled siRNA, or C-Raf siRNA, a 3- to 7-fold reduction in nodule development was observed by cells exposed to siRNA targeting V600EB-Raf (
Fig. 5A and B
; P < 0.05, one-way ANOVA). Knockdown of V600EB-Raf protein expression was confirmed by Western blotting of protein lysates collected following creation of reconstructed skin (
Fig. 5A and C).

siMutB-Raf inhibits melanocytic lesion growth in reconstructed skin. A, targeting melanocytic lesions using siRNA against V600EB-Raf decreases melanocytic lesion development in laboratory-generated skin. The effectiveness of siRNA targeting V600EB-Raf for decreasing cutaneous tumor development was established by nucleofecting GFP-tagged WM35 cells with buffer, scrambled siRNA, or siRNA targeting C-Raf or V600EB-Raf (100 pmol). Cells were then seeded into laboratory-generated skin at time of creation and, 10 d later, average area occupied by green melanocytic lesions was quantified. Top, a statistically significant reduction in green fluorescent lesions was observed following siMutB-Raf treatment (P < 0.05, one-way ANOVA). Columns, mean; bars, SE. Bottom, protein lysates harvested from cells were analyzed by Western blot for B-Raf, C-Raf, pMek1/2, pErk1/2, and cyclin D1 protein expression. Erk2 served as a control for protein loading. B, siRNA-mediated inhibition of V600EB-Raf protein expression in GFP-tagged UACC 903 cells decreases lesion formation in skin reconstructs. UACC 903-GFP cells were nucleofected with siScrambled or siB-Raf (12.5 or 50 pmol) and cells were seeded into laboratory-generated skin at time of creation. Reconstructed skin was analyzed by fluorescence microscopy 10 d later and area occupied by developing GFP lesions was quantified. Columns, mean; bars, SE. C, inhibition of V600EB-Raf decreased MAP kinase signaling in UACC 903-GFP cells. UACC 903-GFP cells were nucleofected with buffer, siScrambled, or siB-Raf (50 pmol) and harvested at 48 h for Western blot analysis. Westerns were probed with B-Raf, pMek1/2, pErk1/2, and cyclin D1 to show decreased MAP kinase pathway signaling. Erk2 served as a loading control. D, mechanistically, siRNA-mediated targeting of V600EB-Raf protein decreased the proliferative capacity of cells. Cultured UACC 903 melanoma cells treated with siMutB-Raf had an increased doubling time, indicating that cells were proliferating at a slower rate (left). Quantifying proliferating cells showed a 2- to 3-fold decrease following siMutB-Raf treatment of tumor cells compared with size- and time-matched tumor controls treated with siRNA to C-Raf (middle and right). Columns, mean; bars, SE.

The mechanism leading to decreased cutaneous tumor development was established by calculating in vitro doubling times as well as comparing differences in proliferation and measuring the effect on MAP kinase signaling. siRNA-mediated inhibition of V600EB-Raf decreased mutant protein expression, causing corresponding decreases in phosphorylated MAP kinase/Erk kinase (pMek) and pErk as well as decreased cyclin D1 expression, indicating reduction in the proliferative signaling pathways of these cells (
Fig. 5A and C). The doubling time of cultured cells containing siRNA targeting V600EB-Raf was found to be ∼1.3-fold higher than that of control cells treated with buffer or scrambled siRNA (
Fig. 5D, left). Furthermore, a 4- to 5-fold decrease in proliferating cells was observed in tumor cells treated with siRNA targeting V600EB-Raf compared with control cells treated with C-Raf siRNA (
Fig. 5D, right). This growth-inhibitory mechanism is consistent with prior reports (
8,
30). Thus, siRNA in nanoliposomes can decrease the expression of V600EB-Raf protein, reducing MAP kinase signaling and the proliferative potential of melanoma cells.

Following ultrasound treatment, siRNA-nanoliposomal complex targeting V600EB-Raf and Akt3 cooperatively decreased melanocytic lesion development in animal skin. The effect of ultrasound followed by topical application of nanoliposomal-siRNA complex on melanoma development in animal skin was examined next. UACC 903-GFP cells (1 × 106) were s.c. injected into the right flank of nude mice and, under anesthesia, treated on alternate days at the tumor site with ultrasound for 15 minutes followed by topical application of siRNA-nanoliposomal complex (
Fig. 6A
). Along with measurement of tumor size (
Fig. 6B), mice were weighed (
Fig. 6B, inset) on alternate days. The effects of liposomes containing siRNA targeting V600EB-Raf or Akt3 alone or V600EB-Raf and Akt3 together on tumor development were compared. Ghost liposomes were added to single treatments to compensate for differences in the amount of liposomal vehicle. A statistically significant ∼30% reduction in tumor size was observed from day 15 in mice treated with complex containing siRNA targeting V600EB-Raf compared with animals exposed to complex containing scrambled siRNA (
Fig. 6B; P < 0.05, two-way ANOVA). In contrast, no statistically significant difference was observed in cutaneous melanoma development following treatment with liposomes containing siRNA targeting Akt3 although treated tumors were 10% to 15% smaller than controls treated with liposomes containing scrambled siRNA (
Fig. 6B; P > 0.05, two-way ANOVA). Significantly, the combination treatment showed cooperatively acting tumor inhibition from day 11 when compared with siScrambled-liposomal complex (
Fig. 6B; P < 0.05, two-way ANOVA).

Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complex alone or in combination with siAkt3-nanoliposomal complex inhibits melanocytic lesion development in animal skin. A, schematic showing treatment regimen. Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complexes that was started the day after injection of melanoma cells and continued on alternate days up to day 23. During the procedure, anesthetized mice were treated with ultrasound at the injection site for 15 min followed by topical application of siMutB-Raf-nanoliposomal complex. B, ultrasound treatment followed by topical application of siAkt3-liposomal complex + siMutB-Raf-liposomal complex decreased melanoma development in animal skin. UACC 903-GFP cells (1 × 106) were injected s.c. into nude mice and, after 24 h, tumors forming at injection sites were treated on alternate days with ultrasound for 15 min followed by topical administration of siMutB-Raf-liposomal complex, siAkt3-liposomal complex, or siAkt3-liposomal complex + siMutB-Raf liposomal complex. Tumors were measured on alternate days beginning on day 3. Control mice were ultrasound treated followed by addition of siScrambled-liposomal complex. Ghost liposomes were added to single treatments so that mice were treated with equivalent amounts of liposomal vehicle. Statistically significant differences between control and siMutB-Raf-liposomal complex + siAkt3-liposomal complex–treated tumors were observed beginning on day 11 (P < 0.05, two-way ANOVA). Columns, mean; bars, SE. Ultrasound treatment followed by topical application of siAkt3-liposomal complex + siMutB-Raf-liposomal complex does not cause a significant change in animal body weight. Animal weights were measured on alternate days beginning on day 1 to determine whether any weight-related toxicity occurred. No significant weight loss was observed between control and experimental groups (P > 0.05, two-way ANOVA; B, inset). Points, mean; bars, SE. C, siMutB-Raf and siAkt3 cooperate to reduce anchorage-independent growth in cell culture. UACC 903 cells were nucleofected with siAkt3 (200 pmol) and siMutB-Raf (1.5, 3, 6, or 12 pmol) in combination and compared with single siRNAs to siAkt3 (200 pmol), siMutB-Raf (1.5, 3, 6, or 12 pmol), siScrambled, or buffer only for the ability to inhibit anchorage-independent growth. D, siAkt3 and siMutB-Raf act additively to inhibit cell viability. Calculation of the combination index (CI) for the combination of siAkt3 and siMutB-Raf showed additive inhibition of cell viability with combination index values between 0.94 and 1.10.

Weights were monitored on alternate days to ascertain weight-related toxicity (
Fig. 6B, inset). No differences were seen in weights, indicating a lack of toxicity (P > 0.05, two-way ANOVA). The lack of systemic toxicity was confirmed by analyzing blood for ALT, AST, alkaline phosphatase, and glucose levels (data not shown), which showed no significant differences between animal groups (P > 0.05, t test). Additionally, no changes in liver morphology were detected in H&E-stained sections (data not shown).

To establish whether growth inhibition would be additive or synergistic following simultaneous inhibition of Akt3 and V600EB-Raf, the Chou-Talalay method was used for determining the combination index using Calcusyn software (
35). Using this approach, combination index values <0.9 are synergistic, >1.1 is antagonistic, and values 0.9 to 1.1 are additive (
Fig. 6D). The estimated combination index values were 0.94, 0.97, and 1.10, for 3, 6, and 12 pmol of B-Raf siRNA plus 200 pmol of Akt3 siRNA, suggesting additive cooperation (
Fig. 6D). Thus, targeting V600EB-Raf and Akt3 in culture or in animals should lead to cooperatively additive inhibition. However, tumor inhibition following liposome-mediated delivery of siRNA targeting V600EB-Raf and Akt3 in
Fig. 6B seems to be more than additive, suggesting that it may tend toward being synergistic.

Discussion

Novel therapeutic regimens need to be developed for treating early or cutaneous metastatic melanoma because, aside from surgical excision, few treatment options are available (
39). Current systemic therapeutics cause toxicities and have off-target effects (
19). Topical or localized treatments with agents could permit use of high local concentrations with minimal toxicity and be useful for treating cutaneous lesions not amenable to surgical removal (
19,
40). A case in point is the use of interlesional injections of interleukin 2 (IL-2) and electroporation for improved topical delivery of bleomycin for melanoma treatment (
40,
41). Effective topical agents could provide additional, currently unavailable, treatment options for patients with early or invasive cutaneous melanoma.

The era of personalized, targeted cancer treatment is near, but specific proteins playing key roles in particular cancer types need identification and the demonstration that simultaneous targeting inhibits tumor development in a synergistic manner. This study shows that V600EB-Raf and Akt3 are two such targets in melanoma, the inhibition of which led to cooperative tumor inhibition. Furthermore, a novel approach has been developed for delivering therapeutic siRNA into early or invasive cutaneous melanocytic lesions targeting these important proteins. Ultrasound treatment of skin followed by topical application of nanoliposomes containing siRNA targeting V600EB-Raf and Akt3 reduced the expression of these proteins, significantly decreasing melanoma development in a cooperatively additive manner.

Simultaneous inhibition of the MAP and phosphatidylinositol 3-kinase pathways has previously been reported to enhance the inhibitory effect on both signaling cascades leading to increased tumor cell apoptosis, which supports the feasibility of this approach (
42–
44). Furthermore, siRNA-mediated inhibition of V600EB-Raf and Akt3 in aggressive melanoma cells has been shown to cooperatively inhibit tumor development through enhanced effects on the signaling cascades, specifically leading to decreased cyclin D1 expression as well as increased p27 and cleaved caspase-3 levels (
9). These results are not surprising considering discoveries linking the two pathways in early melanoma development (
9). Approximately 90% of nevi express V600EB-Raf, but very few ever develop into melanoma due to the inhibitory effects associated with high MAP kinase pathway activity caused by the mutant protein. Akt3 has been shown to overcome this block by phosphorylating V600EB-Raf in these melanocytes to lower the activity of mutant protein and downstream MAP kinase pathway signaling to levels promoting, rather than inhibiting, melanoma development (
9). Phosphorylation of V600EB-Raf enabled melanocytes to overcome the growth-inhibitory effects associated with high pathway activity and to develop melanoma cell–like characteristics (
9). Thus, this discovery shows that siRNA can be used as a therapeutic specifically targeting multiple aberrantly expressed or mutant cancer-causing genes without inhibiting the activity of normal proteins to more effectively treat melanoma.

Systemic administration of free unmodified siRNA by i.v. injection is not clinically feasible because it is rapidly degraded by serum factors, preventing clinically useful concentrations from being reached (
45). Development of novel vehicles for loading, protecting, and delivering siRNA to distant sites around the body is one strategy (
16–
18,
46,
47). This study shows that cationic nanoliposomes can load, protect, and deliver therapeutic siRNA into melanoma cells. Nanoliposomes are taken up by all cell types but only affects those containing V600EB-Raf or overexpressing Akt3. The specificity of siRNA for its target reduces toxicity and potential side effects. Furthermore, use of pegylated lipids and small average nanoliposomes with a diameter of ∼50 nm decreases immunologic or inflammatory responses, enabling the particles to bypass the reticulo-endothelial system (
48,
49).

Ultrasound was used to permeabilize skin, enabling delivery of siRNA-liposomal complex throughout the epidermal and dermal layers because it had been reported to be effective for delivering various macromolecules into and throughout skin (
19,
21–
26). A lightweight transducer with shown capabilities of delivering insulin or antisense oligonucleotides into or through skin was used, showing that low-frequency ultrasound can permeabilize skin, enabling delivery of nanoliposomal-siRNA into melanoma cells residing at the dermal-epidermal junction to inhibit protein expression and melanoma development.

Mechanistically, a therapeutic nanoliposomal-siRNA complex targeting V600EB-Raf and Akt3 can effectively decrease the expression of both proteins and downstream signaling cascades to inhibit the development or spread of cutaneous melanoma (
8,
10,
30). Thus, a nanoliposomal-siRNA complex targeting these proteins could be used as a nonsurgical approach to decrease the number of benign moles or inhibit early cutaneous melanocytic lesion development (
2–
5,
9). The latter would have the consequence of decreasing the spread at the earliest stages, thereby prolonging survival by preventing metastases. Ultrasound in conjunction with a therapeutic topical nanoliposomal-siRNA complex targeting V600EB-Raf and Akt3 could also provide a treatment option for patients whose disease is not amenable to surgical removal or limb perfusion when widespread cutaneous lesions are present on the trunk. No therapeutic agents for treating these patients are available. Furthermore, advanced-stage melanoma patients could benefit by administering these agents at the time of sentinel lymph node biopsy or surgery to target cells that may not have been removed during the procedure (
50). Finally, this approach could be combined with more traditional therapeutic agents to improve clinical efficacy through synergistically acting mechanisms.

In conclusion, topical delivery of cationic nanoliposomes loaded with siRNA targeting V600EB-Raf and Akt3 in combination with low-frequency ultrasound has potential to decrease early melanocytic lesion development in skin or prevent the spread of cutaneous melanoma metastases.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank James Kaiser, Julie Anderson, Nishit Trivedi, Mitchel Cheung, and SubbaRao V. Madhunpantula for technical assistance, and Shantu Amin for critical reading of the manuscript.